Cancer is the second leading cause of death after heart disease in the US. Chemotherapy is a mainstay of treatment after surgical removal of tumors; but the balance of clinical benefit versus disabling or life- threatening side effects is often uncertain. Genotyping of cancers to identify mutated oncogenes has enabled an era of targeted therapy. Drugs targeting the mutated proteins that drive tumor growth promised to revolutionize cancer treatment; but the genetic plasticity inherent in cancer limits the numbers of patients who can respond to treatment, and those that do, often relapse due to development of drug resistance. This proposal describes prodrugs intended to kill tumors with reduced damage to healthy tissues. The prodrugs are designed to remain harmless until they are cleaved by the enzyme fibroblast activation protein (FAP). Short peptides are linked to cytotoxic molecules (tumor-killing warheads) to create prodrugs that only release their warheads when a specific peptide bond is cleaved enzymatically by FAP. FAP is expressed by nonmalignant fibroblasts in the connective tissue (stroma) of epithelial tumors; therefore, prodrugs enable tumors to be targeted with cytotoxic agents independently of the mutational status of the cancer cell. Prodrug feasibility was demonstrated in STTR Phase I for ARI-3996, which delivers a Velcade-like proteasome inhibitor to the tumor, and confirmed with ARI-3099DOX, which delivers the chemotherapeutic agent doxorubicin (DOX). ARI-3099DOX and ARI-3996 are both promising drug candidates. Before proceeding to IND-enabling studies, however, further work, which is planned for STTR Phase II, will be required in order to: (1) improve prodrug half-life in vivo, (2) evaluate the possible safety risk that might result from killing FAP+ cells that have recently been discovered in normal tissues, and (3) understand whether, by a new mechanism of action, prodrugs can relieve tumoral immune suppression to activate the immune system to kill tumors [1]. Arisaph has developed chemistry required to make prodrugs that are unique in that they are cleaved to release cytotoxic warheads by FAP, but not by a closely related enzyme, prolyl endopeptidase, which would otherwise present a major risk of toxicity to the patient because it is expressed in many healthy tissues. Developmental risk is mitigated by ability to make back up compounds, and clinical risk, by patient selection with a simple biopsy assay for FAP activity in tumor samples. Arisaph's collaborator, Dr. H. Borghaei (Fox Chase Cancer Center), has developed a model of endogenous lung cancer for testing the possible immunological effects of prodrugs. The goal of STTR Phase II is to select the most efficacious prodrug candidate, based on preclinical efficacy and safety, for IND-enabling studies that will be conducted by Arisaph in Phase III.